This project targets HIV-1 envelope protein and consists of a novel approach to the identification and development of envelope proteins that can induce highly cross-clade reactive, primary virus neutralizing antibodies (NA) when expressed in vivo using Venezuelan equine encephalitis virus (VEE) replicons. A clade B env gene will be used that was obtained from a patient with a broadly cross-reactive, primary virus NA response. This env gene expresses a protein, designated R2, with epitopes that, when expressed on pseudoviruses (PV), cross-react with NA from people infected with other clades of HIV. The use of PV technology for the evaluation of neutralization phenotype of env genes already has been extensively evaluated in the laboratory of the Principal Investigator. This technology should permit the Principal Investigator to engineer the gene to alter its phenotype in systematic ways, and to test the relationship between structure and immunogenicity. The extent of the cross-reactivity of R2 will be further defined using PV expressing env of various clades and sera from people infected with different clades. Envelopes will be sought which cross-react among sera that do not appear to group antigenically with R2 to be used in comparative in vitro and immunogenicity studies. The epitopes responsible for the cross-reactivity of R2 will be sought by competitive binding studies using sera with NA which do and do not neutralize R2 and monoclonal antibodies which are currently available, as well as some to be developed against R2 in this project. Epitope mutagenesis will be performed to further define the nature of the cross-reactive epitopes and, potentially to attempt to introduce epitopes with distinct spectra of cross reactivity. The influence of conformation on function and immunogenicity of specific epitopes will be studied by introduction of targeted conformation altering mutations. These will be targeted on the basis of principles learned from ongoing studies of gp120-gp41 interaction. Immunogenicity studies will define an appropriate immunization regimen using the VEE replicon system, and comparative studies will be performed to attempt to identify proteins which induce distinct spectra of highly cross-reactive, primary virus NA.